Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

Abstract

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Keywords: Chlamydia; apolipoprotein; cell-free expression; major outer membrane protein; membrane protein; nanolipoproteins; nanotechnology; oligomer; telodendrimer.

Figure 1.

mMOMP–tNLP preparation. a–c, mMOMP DNA, Δ49ApoA1 DNA, and DMPC/telodendrimer were mixed in a cell-free reaction chamber. d, protein translation and the self-assembly of mMOMP–tNLPs in a cell-free lysate. e, the assembled mMOMP–tNLP product is shown.

Figure 2.

Codon-optimized sequences. DNA sequences of Δ49ApoA1 (a) and mMOMP (b) proteins are shown with nucleotide alterations in white background. All mutations are silent mutations.

Figure 3.

Expression and purification of mMOMP–tNLP. a, SYPRO Ruby protein gel stain of 4–12% SDS-PAGE of affinity-purified mMOMP–tNLP. Total cell-free lysate (Total), flow-through (FT), Washes 1 and 6 (W1 and W6), and Elutions 1 through 6 (E1–E6) are shown. All samples were boiled for 5 min in the presence of 50 mm DTT. mMOMP is at 40 kDa, and the Δ49ApoA1 is at 22 kDa. M, molecular mass marker. b, SEC traces of mMOMP–tNLP particles. Different elutions still maintain the same sized mMOMP–tNLPs. c, dot blot of mMOMP–tNLP sample (SEC peak fraction using the nickel affinity purified elution E2 as input) in duplicate probed with mAb40 and mAb-His.

Figure 4.

mMOMP insertion increases the size of tNLPs. a, the size distribution of empty tNLPs as measured by DLS. b, the size distribution of mMOMP–tNLPs measured by DLS. c, cryoEM demonstrates that the mMOMP particles are disc-like in shape and that there are size differences between tNLP and mMOMP–tNLP particles. High-density areas can be seen in the images of mMOMP–tNLPs, indicating pore formation. Scale bars, 10 nm.

Figure 5.

mMOMP forms higher order structures in mMOMP–tNLPs by SDS-PAGE and Western blotting. a, SYPRO Ruby protein gel stain of 4–12% SDS-PAGE of affinity-purified mMOMP–tNLP and tNLP alone. mMOMP monomer shows a band at ∼40 kDa, and the Δ49ApoA1 shows a band at 22 kDa. The mass ratio of soluble mMOMP to apolipoprotein is ∼2:1 as determined through densitometry analysis. In lane 2, heat and reducing agent break down higher order mMOMP structure. In lane 3, the presence of higher order bands indicates mMOMP multimer conformation. b, Western blot of mMOMP–tNLP and tNLP alone. Shown is a transfer membrane probed with mAb40 (1:1,000 dilution). As indicated, samples were incubated at room temperature or boiled for 5 min in the presence of 50 mm DTT. M, molecular mass marker.

Figure 6.

mMOMP–tNLPs initiate pore-forming channels. a, conductance traces recorded at 50-mV applied voltage in physiological conditions after tNLP alone (trace i) and mMOMP–tNLPs (traces ii–iv) were added to the measurement chamber. Current increases seen after mMOMP–tNLP addition indicate pore formation. Representative traces show 1× (trace ii) and 3× (trace iii) mMOMP–tNLP incorporations. Trace iv shows several incorporations events occurring in quick succession. b, histogram of 184 conductance events. The dashed line indicates the best fit to a sum of Gaussian peaks for 1× and 3× incorporation events. pS, picosiemens.

Figure 7.

In vivo testing with mMOMP–tNLPs. a, mMOMP–tNLPs elicit significant antibody titers. Sera from mice immunized with mMOMP–CpG–tNLPs, CpG–tNLPs, and PBS were loaded on ELISA plate precoated with mMOMP–tNLPs (black dots) or empty tNLPs (red dots), and antibody titers were measured. b, four Western blots are shown. The mMOMP protein is loaded onto each lane equally. Mouse sera from the mice immunized with the four different conditions were then incubated with the blot overnight. Lane 1 is blotted with mouse sera immunized with mMOMP–CpG–tNLP. This blot shows significant mMOMP antibody binding. Lane 2 is blotted with mouse sera immunized with empty tNLP–CpG. Lane 3 is blotted with mouse sera immunized with live Chlamydia EB. This blot confirms that Chlamydia EB induces MOMP antibodies that bind to our recombinant mMOMP. The decreased signal from this blot is because Chlamydia EB contains many surface antigens, not just mMOMP; therefore, it induces a large variety of antibodies. Lane 4 is blotted with mouse sera immunized with PBS control group and shows no mMOMP binding. M, molecular mass marker.